Biotechnology: Using living systems to solve problems

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Fats and oils play an important role in many aspects of everyday life. Having the right balance of fatty acids in our diet is important to health, not to mention the countless products that contain oils and their derivatives as key ingredients.

As the earth’s population continues to grow, the need for fats and oils will likewise increase. In anticipation, oilseed researchers are preparing themselves to meet the growing demands for fats and oils.

One key way forward will involve the use of biotechnology to generate new oilseed crops that have improved fatty acid content and yields, for edible, personal care, and industrial applications. This article provides an overview of what biotechnology is and how the tools and techniques apply to the fats and oils industry.

For many millennia, humans have exploited biological systems to create useful products. Long before the underlying biology was understood, people have used yeast to create bread and wine, mold to create aged cheeses, bacteria to ferment cucumbers into pickles, and microbes to perform the retting process that converts flax into linen. If we apply Merriam-Webster’s definition, these are among the earliest examples of biotechnology, which, in the broadest sense, involves “the use of living cells, bacteria, etc., to make useful products.”

The applications of biotechnology in agriculture date back hundreds of years to when farmers began growing crops in new environments, such as the introduction of soybean to North America from China in the 18th century. They used traditional breeding methods, crossing plants with certain desirable features to create new varieties with traits such as disease resistance and tolerance to pests, chemicals, and extreme environments. Although the underlying biology was not understood at the time, this was the beginning of human intervention resulting in genetic alterations to crops.

Modern biotechnology began with the first demonstration of genetic engineering (GE) in the 1970s, when scientist Paul Berg carried out the first successful gene splicing experiment. Shortly after, researchers Herbert W. Boyer and Stanley N. Cohen transferred genetic material into a bacterium and demonstrated that the new genes were reproduced along with the organism’s native DNA. Since then, scientists have used GE to render crops resistant to pests, herbicides, and drought conditions, and to improve their nutrient profiles. In addition, GE has led to innovations in medicine, biofuels, and bioremediation.

How genetic engineering works

The two most common approaches to creating GE plants involve the use of recombinant DNA technology and can be broken down into two basic steps. First, researchers identify and isolate a gene of interest, then they introduce that gene into plant cells to create a transgenic, or GE plant.

In one method, researchers recruit the natural soil bacterium Agrobacterium tumefaciens and replace some of its genes with those encoding the desired trait. The bacterium is then allowed to infect a plant, during which it transfers its DNA into the plant’s genome in a process biologists refer to as transformation. The result is a new, genetically modified plant, also known as a transgenic crop.

Another approach involves attaching the DNA of interest to the surfaces of gold or tungsten microparticles. Researchers then use an instrument known as a biolistic gene gun to blast the particles into a plant’s cells. This method is also known as microprojectile bombardment. Some of the DNA will get inserted into the genome through a process known as homologous recombination (Biocat. Agric. Biotech., 3:31-37, 2014).

These transformation methods have been described as “brute force” methods owing to their high failure rates, which require numerous attempts before a candidate for a commercial product is identified. According to a recent survey of agricultural biotechnology developers, more than 10,000 genes are evaluated on average for each commercial product (http://tinyurl.com/PhillipsMcDougallStudy). The evaluation includes bioinformatics assessments, which involve the use of computers to compare genetic sequences, and helps ensure the safety of GE crops by eliminating sequences that are genetically similar, or homologous, with known allergens and toxins. Bioinformatics can also help predict whether the gene will have the desired function, according to a review article in the Journal of Agricultural and Food Chemistry by Laura S. Privalle and coworkers (61:8260-8266, 2013).

Out of those 10,000 genes, roughly 500 are selected for proof-of-concept experiments. Then, more than 1,000 transgenic organisms are further evaluated, as described below. Finally, one or two of these organisms are chosen for commercialization. Not surprisingly, the process of developing a single commercialized GE product comes with significant costs of both time and money, averaging about 13 years and $150 million. (See sidebar: “Interesting stats about genetically engineered crops.”

Evaluating a GE crop candidate

In order to select the most promising GE crop candidates from the hundreds of transgenic organisms created, researchers perform numerous tests. Before a candidate can proceed, it must demonstrate that the gene was inserted at a location that does not disrupt essential cellular functions, that the inserted gene is expressed at desirable levels, and that the desired trait is present and passed on to progeny. The GE plant must also be evaluated for how well it performs as a crop: Is the yield adequate, and is the desired phenotype—the characteristic the plant was engineered to have—observed? Simply having the correct genotype—or genetic composition—does not guarantee the intended characteristic will show up in the organism, since environmental and developmental conditions can play a role in gene expression (J. Ag. Food Chem., 61:8260-8266, 2013).

The vast majority of the transformed organisms created will have undesirable insertion sites, or one or more other unfavorable properties, which is why researchers work with large sample sizes at this stage.

On to commercialization

Once a candidate for a commercial crop is selected, the species undergoes additional analyses, as described by Privalle and coworkers, (J. Ag. Food Chem., 61:8260-8266, 2013) to determine “the safety of the protein produced by the gene, plant performance, the impact of cultivating the biotechnology crop on the environment, agronomic performance, and the equivalence of the crop/food to conventional crops/food.”

Part of the safety evaluation involves a compositional analysis, in which researchers do a side-by-side comparison of the nutrient and antinutrient content of the GE crop compared to its parental line, as well as other conventional, or non-transgenic, lines. The purpose of a compositional analysis is to determine whether the GE crop is substantially equivalent to a crop that has a history of safe use. The specifics of the compositional analysis and other safety assessments depend on the regulatory standards set by the country in which the GE crop will be marketed (J. Ag. Food Chem., 61:8260-8266, 2013) but typically include studies on the mode of action, expression levels, toxicity, and allergenicity of the inserted trait. More details regarding the general guidelines for safety assessments can be found in the 2013 Codex Alimentarius Commission (http://tinyurl.com/CODEX-rDNA-plants).

Concern over impacts of GM crops on the environment and health

In their 2014 review article, Maheshwari and Kavolchuk highlight some of the public’s major concerns over the rising use of GE crops worldwide (Biocat. Agric. Biotech., 3:31-37, 2014).

In many parts of the world, the use of GE crops is controversial and contested: “A fear of the unknown dangers associated with the technique and underlying risk factors have been a strong concern,” the authors write. “The main concerns include food safety, the transfer of genetic material from transgenic plants to other species, including other plants, microbes, animals, a cumulative effect on ecosystems and biodiversity, the corporate control of the food and seed supply, and other moral/religious/ethical concerns.”

The risks associated with GE crops are dependent on the function of the inserted gene, they write. “Genetic engineering is still an imprecise technology, and the random incorporation of genes might lead to unpredictable disturbances in the plant genome, physiology and biochemistry… But to date, there is no clear evidence that genetically engineered crops are harmful.”

To safeguard against unknown health hazards, Maheshwari and Kovalchuk recommend researchers “develop a battery of tests for the analysis of potential toxicity, mutagenicity and allergenicity of [GE] crops.”

Alan McHughen, a molecular geneticist who is actively involved in public engagement on the topic of GE foods, shared his advice on addressing questions from the general public at the 2014 AOCS Meeting. See: “Addressing common myths about genetically engineered organisms.”

Genetically engineered oil crops

Several decades of research in seed oil biosynthesis resulted in a general understanding of which genes are involved in oilseed modification, which set the stage was for “the genetic engineering of oilseed crops that produce designer plant seed oils tailored for specific applications,” according to J.M. Dyer and R.T. Mullen in a 2005 review in Seed Science Research (15:255-267, 2005). “Although traditional breeding methods have been used successfully to alter the fatty acid composition of oils,” they wrote, “GE provides a more rapid and direct method for manipulating fatty acid composition and can greatly expand the types of fatty acids produced in oil seeds.”

Among the many important research avenues involving GE crops is the development of oilseeds with valuable traits such as increased vitamin content, higher levels of healthy fatty acids, tolerance to unfavorable growing conditions, such as drought and various biotic and abiotic stresses (Biocat. Agric. Biotech., 3:31-37, 2014). Table 1 contains a list of the top producers of major oilseed crops worldwide, and Table 2contains a sampling of GE soybean crops that have been modified to increase the production of certain desirable fatty acids.

Another important area of GE oilseed research is for the development of oilseed crops that produce oils not normally found in plants. Table 1 contains a list of the top producers of major oilseed crops worldwide, and Table 2contains a sampling of GE soybean crops that have been modified to increase the production of certain desirable fatty acids.

Industry researchers can take comfort in knowing that the composition and quality of oil derived from GE crops will be the same as oils derived from conventional crops, according to Susan Knowlton, senior research manager at DuPont. There’s “virtually no difference” between the processes for producing oils, whether they’re derived from GE or non-GE crops, Knowlton said. “Any oil that’s gone through standard refining, bleaching, and deodorizing operations” will be free of DNA, proteins and residual chemicals, such as pesticides.

Table 1: Major oilseed crop production varies across the globe. Data represent averages from the 2012-2013 growing season. Source: USDA World Crop Production Summary.

Table 2: For nearly two decades, researchers have been using the tools of genetically engineering to create crops with higher levels of desired fatty acids. Here’s a sampling of GE soybean crops. Adapted from: Biocat. Agric. Biotech., 3:31-37, 2014

Major technical challenges to genetically engineered oil crops

Although GE oil crops have been around for 20 years, there is still much room for improvement, which will require a better understanding of the biochemical processes underlying fatty acid biosynthesis, according to Maheshwari and Kavolchuk.

Because the “seed oil content of plants is controlled by multiple steps in the oil biosynthetic pathway,” they write, it is unrealistic to expect the insertion of a single gene will result in a dramatic increase in oil yield. To boost yields, not only do the right genes need to be present, the proteins they encode need to be expressed at the proper levels, in the proper tissues, and at the proper time. But there are a few simple strategies involving single gene modifications that hold promise for improving oil content, such as down-regulating genes to reduce levels of unwanted fatty acids, or overexpressing other genes to increase the desired ones. (See Table 2).

Once a GE oil seed is created, researchers must also insure that (1) the oils stored in the seeds don’t interfere with the germination process, resulting in unviable seeds, (2) the transgenes are expressed at optimal levels, and (3) that the oils produced aren’t toxic to the plant itself and don’t interfere with other cellular processes (Biocat. Agric. Biotech., 3:31-37, 2014).

Up-and-coming techniques for editing DNA

The two commonly used approaches to creating GE crops—Agrobacterium tumefaciens and microprojectile bombardment—share one major downside: Both techniques cause the gene-of-interest to insert into the host’s DNA at random, which can result in the disruption and/or truncation of native genes.

To address this shortcoming, researchers have been working to develop other transformation methods that would scientists to determine the exact spot in which a gene will be inserted.

Here’s a sampling of some of the up-and-coming techniques for genome editing:

Clustered regularly interspaced short palindromic repeats (CRISPRs) are DNA molecules, inspired by naturally occurring CRISPRs found in bacteria and archaea, that can be designed to insert into an organism at any desired location (Nat. Rev. Genetics, 11:181-190, 2010).

Transcription activator-like effector nucleases (Nat. Biotech, 29:135-136, 2011) and zinc-finger nucleases (Gen. Soc. Am., 188:773-782, 2011) belong to the family of enzymes known as artificial restriction enzymes. They contain two primary components: a DNA binding domain, which recognizes a specific DNA sequence, and a DNA cleavage domain, which cuts the DNA to enable the gene-of-interest to insert into the genome at a pre-determined location.

The future of genetic engineering

The biggest push so far for commercialized GE oilseed crops thus far has been for herbicide- and pest-resistant crops, which directly address farmers’ needs. But some experts expect the industry will soon be making a shift toward crops that directly meet consumers’ needs and demands, such as with the creation of oils that have more healthful fatty acid profiles. “That story’s just occurring now,” Knowlton said.

Another important research avenue for GE oilseed crops involves engineering “metabolic pathways for production of exotic fatty acids into more traditional oilseed crops,” wrote Dyer and Mullen (Seed Sci. Res., 15:255-267, 2005). A plant with the ability to create oils normally produced in fish, such as the widely popular omega-3 fatty acids DHA and EPA, would find enormous value among both industry and consumers. As a step in this direction, researchers at Monsanto have announced plans to commercialize a GE soybean oil rich in stearidonic acid, which the body can convert into EPA, an omega-3 fatty acid that may help maintain heart health (Lipids, 43:805-811, 2008).

Other efforts have been made to increase the amount of oil content present in a plant’s leaves, which could lead to the development of new sources for biofuels (inform, 23:206-210, 2012). In one study, researchers engineered plants to generate six times more lipids in their leaves than their non-engineered counterparts (Plant Biotechnol. J., 9:874-883, 2011).

The biggest challenge in the field of GE crop development, however, will be winning the public’s trust regarding GE foods. Without consumer demand, Knowlton explained, companies have no guarantee that they will be able to make up the cost of producing GE crops specifically designed with their needs in mind. “It’s an expensive technology… and [companies] have to have some assurance in the end that there’s a payback on the investment,” she said. But “until consumers recognize that things can be done to address and improve their lives specifically, it’s hard for them to readily adopt the technology.”

Knowlton believes that educational initiatives like GMO Answers (http://gmoanswers.com/), a website that allows people to submit questions and receive answers from biotech supporters including scientists, farmers and science communicators, will help win over the public for GE foods. “There’s a very bright future,” she said, “because a lot of changes, certainly in the fats and oils space, can be made providing things like omega-3s [and other healthful fatty acids] in oils. These are all changes that are technically doable, but you need the consumer acceptance to roll them out.” GMO Answers, which is funded by members of The Council for Biotechnology Information, which includes BASF, Bayer CropScience, Dow AgroSciences, DuPont, Monsanto Company, and Syngenta, highlights the vast potential of GE crops to increase crop yields, maximize land mass and water resources, and provide food to an ever-increasing global population. The success of such initiatives, which are still in their early stages, are yet to be determined.

While web-based educational initiatives that address public concerns on a large scale are important, McHughen believes every scientist has a responsibility to speak up and engage with the people in their sphere of influence on the topic of GE foods (for McHughen’s advice about public engagement, see “Addressing common myths about genetically engineered plants.” Although it can be extremely frustrating at times, he said, “it can be very rewarding when you talk to people who say, ‘Thank you, I’ve learned so much.’” And it may be the only hope of realizing the full potential of GE crops for the benefit of the world.

Sidebar

Glossary of agricultural biotechnology terms

Biotechnology: The use of biological systems to create useful products.

Traditional plant breeding: An age-old practice that involves selecting and breeding, or crossing, plants to yield new crops with valuable traits, including increased quality and yield, as well as disease resistance and tolerance to pests, chemicals, and extreme environments.

Genetic engineering (GE): A modern agricultural biotechnology tool that involves altering the genetic makeup of an organism using recombinant DNA technology (rDNA). GE may involve the transfer of genes between species, which distinguishes it from traditional breeding. GE crops are also described as transformed, transgenic, or genetically modified.*

Transformation: A stable genetic change resulting from a cell incorporating exogenous, or non-native, DNA into its genome. If the process occurs in animal cells, it is known as transfection.

Transgene: A gene that is transferred from one organism to another, as a result of either traditional breeding or GE.

Nucleotides: The individual chemical units that make up DNA and RNA.

Splice: The process of combining fragments of DNA to create genetic material known as recombinant DNA.

Homologous recombination: The process of exchanging nucleotide sequences between two similar DNA molecules.

Recombinant DNA technology (rDNA): A technique that involves the use of enzymes to cut out, multiply, alter, and insert pieces of DNA into an organism.

Genotype: The genetic makeup of an organism.

Phenotype: The observable traits of an organism.

Bioinformatics: The use of computers to compare the genetic information. When assessing GE crops, scientists use bioinformatics to avoid inserting genes that have similarities with known allergens or toxins.

Agronomic performance: How a crop behaves in the field, including factors such as yield, composition, and other desired traits.

Compositional analysis: Performed by crop scientists to determine whether GE crops are equivalent to conventional crops based on an assessment of nutritional value.

Footnote: *GE crops are often referred to as genetically modified organisms (GMOs), but since traditional breeding techniques also result in genetic modification, the more precise term to describe the method used to create new organisms through rDNA technology is genetic engineering.

Across the globe, 18 million farmers grow GE crops in 27 countries on roughly 400 million acres of land, according to the International Service for the Acquisition of Agri-biotech Applications (ISAAA). The top growers of GE crops are (in descending order): the U.S., Brazil, Argentina, India, and Canada.

Across the globe, roughly 80% of all soybean and cotton, and 35% of corn, is GE. In the U.S., about 90% of all corn, cotton, canola, sugar beet, and soybean is GE.

The average GE crop take about 13 years and $150 million to produce, from start to finish, according to Cynthia Ludwig, a former scientist and communicator for Monsanto Company (U.S.), which is the leader in the global proprietary seed market.

Sidebar

Addressing common myths about genetically engineered plants Alan McHughen is a professor of molecular genetics at UC Riverside and author of the book “Pandora’s Basket: The Potential and Hazards of Genetically Modified Foods.”

McHughen spoke at the 2014 AOCS National Meeting on the topic of GMO policy, where he offered some advice to scientists on how to engage with the public on this controversial topic. The public has numerous misconceptions about GE. But everyday scientists can—and should—help relay some of the public’s fears, by helping them understand a few basic concepts:

The process of genetic engineering, in and of itself, is not harmful. The merits of each GE product must be evaluated individually rather than assuming that because a product was made with GE techniques it is automatically unsafe.

Scientific studies overwhelmingly show the safety of GE foods. Numerous national and international health organizations, including the World Health Organization, National Academy of Sciences, and the American Association for the Advancement of Science, declare that the benefits of GE foods outweigh the risks.

Just because GE is “unnatural” does not mean that it’s unsafe. There are plenty of naturally occurring chemicals and organisms that are extremely dangerous.

The dose makes the toxin. A large dose of an otherwise harmless substance can be deadly, such as the nutrient Zinc, while low doses of chemicals, such as the pesticide glyphosate (Roundup), can be safe.

Correlation does not equal causation. “I hear people say GMOs were introduced in the mid-90s, and they’ve increased the same rate as autism,” McHughen said. Using that logic, one could argue that organic food sales, which also correlate with autism rates, are the cause of autism, he said.

Oils derived from both GE and conventional oilseeds are processed to remove DNA and proteins, so they are compositionally identical (unless they’ve been intentionally altered to have a different fatty acid composition).